The present disclosure relates to cellular analysis techniques, and more particularly, to systems and methods for in situ laser lysis for analysis of biological tissues (live, fixed, frozen or otherwise preserved) with single cell resolution.
Cells in living tissue have heterogeneous responses to environmental cues because of their differences in cell types, locations, exposure to blood supply, malignancy, and/or infection. For instance, cell-cell communication between cancer cells and their environments at the primary tumor and distant metastasis sites have been shown to be important for cancer development (Zhang W, et al. 2011, Cancer biology & therapy 11: 150-156; Calorini L, et al. 2010, Cell communication and signaling: CCS 8: 24). Recent technological advances in assessing gene expression at the single cell level have enabled advancements in the way in which investigators study diseases. The analysis of gene expression can be performed on live, preserved, and frozen tissues.
The introduction of commercially available microfluidic high-throughput systems further enables researchers to investigate problems at a larger scale (Citri A, et al. 2012, Nature Protocols 7: 118-127; Guo G, et al. 2010, Developmental Cell 18: 675-685). However, such methodologies may require cells to be dissociated from their native environments and therefore, may obscure valuable biological states that are influenced by multicellular complexity in situ. Current tools are not able to capture lysate from individual cells in situ, especially making it difficult to analyze the individual live cells given the short-lived nature of RNA, which can degrade on time scales of seconds to minutes, as well as the fast response time of the cellular gene-expression machinery on the order of minutes.
One single-cell lysate harvesting approach includes a vacuum-like mechanical probe to continuously release lysis buffer through a microchannel and draw in liquid surrounding target cells (Sarkar A, et al. 2014, Nature Communications 5). However, the size of the probe head resulted in a physical limitation with respect to accessing the target cells. Moreover, the time required for cell lysis is relatively lengthy at around 1 minute. Finally, harvesting a quantity of cells to acquire statistically significant results can be in excess of one hour, which may trigger cellular stress responses to the surrounding cells. Accordingly, it would be useful to provide a system and method for single cell analysis which can be accomplished under biologically relevant conditions and on biologically relevant time-scales.